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The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model

View ORCID ProfileRobert C. M. Knaap, View ORCID ProfileRaúl Fernández-Delgado, Tim J. Dalebout, View ORCID ProfileNadia Oreshkova, Peter J. Bredenbeek, View ORCID ProfileLuis Enjuanes, View ORCID ProfileIsabel Sola, View ORCID ProfileEric J. Snijder, View ORCID ProfileMarjolein Kikkert
doi: https://doi.org/10.1101/751578
Robert C. M. Knaap
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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Raúl Fernández-Delgado
Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain
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Tim J. Dalebout
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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Nadia Oreshkova
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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Peter J. Bredenbeek
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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Luis Enjuanes
Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain
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Isabel Sola
Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain
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Eric J. Snijder
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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Marjolein Kikkert
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
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  • For correspondence: m.kikkert@lumc.nl
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Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause zoonotic infections and serious disease, primarily in the Arabian Peninsula, due to repeated spill-over from dromedary camels and subsequent nosocomial transmission. Approved MERS vaccines for use in animals or humans are not currently available. MERS-CoV replication requires the virus-encoded papain-like protease (PLpro) to cleave multiple sites in the viral replicase polyproteins, thereby releasing functional non-structural proteins. Additionally, PLpro is a deubiquitinating enzyme (DUB) that can remove ubiquitin(-like) moieties from substrates, presumably to counteract host antiviral responses. In previous work, we determined the crystal structure of MERS-CoV PLpro in complex with ubiquitin, facilitating the design of PLpro mutations that impair DUB activity without affecting viral polyprotein cleavage. Here, we introduced these DUB-inactivating mutations into the viral genome and examined their impact on MERS-CoV infection both in cell culture and in a lethal mouse model. Although overall replication of DUB-negative and wild-type (wt) recombinant MERS-CoV was comparable in multiple cell lines, infection with DUB-negative virus markedly increased mRNA levels for interferon (IFN)-β and IFN-stimulated genes. Moreover, compared to a wt virus infection, the survival rate was significantly increased when DUB-negative MERS-CoV was used to infect transgenic mice expressing a human MERS-CoV receptor. Interestingly, DUB-negative and wt MERS-CoV replicated to the same titers in lungs of infected mice, but the DUB-negative virus was cleared faster, likely due to the observed accelerated and better-regulated innate immune responses, in contrast to delayed and subsequently excessive responses in wt virus-infected mice. This study provides the first direct evidence that the DUB activity of a coronaviral protease contributes to innate immune evasion and can profoundly enhance virulence in an animal model. Thus, reduction or removal of the innate immune-suppressive DUB activity of PLpros is a promising strategy for coronavirus attenuation in the context of rational vaccine development.

Author Summary Although zoonotic coronaviruses such as Middle East respiratory coronavirus (MERS-CoV) have pandemic potential, therapeutics and vaccines that counteract this public health threat are not currently available. Coronaviruses typically employ multiple strategies to evade the host’s innate immune response, which may enhance clinical disease and/or reduce the efficacy of modified live vaccines. The MERS-CoV-encoded papain-like protease (PLpro) is not only crucial for the expression of functional replicase proteins, but has also been postulated to antagonize ubiquitination-dependent steps during the activation of the innate immune response. Here, we report the generation of engineered MERS-CoVs mutants in which PLpro’s deubiquitinating (DUB) activity was specifically disrupted without affecting virus viability. In this manner, we could demonstrate that the DUB activity of PLpro suppresses the interferon response in MERS-CoV-infected cells. Strikingly, in the lungs of mice infected with DUB-negative MERS-CoV, innate immune responses were induced at an earlier stage of infection than in wt virus-infected mice. This group also showed a clearly increased survival, indicating that the DUB activity is an important MERS-CoV virulence factor. This proof-of-concept study establishes that the engineering of DUB-negative coronaviruses, which elicit a more effective immune response in the host, is a viable strategy for vaccine development.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 29, 2019.
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The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model
Robert C. M. Knaap, Raúl Fernández-Delgado, Tim J. Dalebout, Nadia Oreshkova, Peter J. Bredenbeek, Luis Enjuanes, Isabel Sola, Eric J. Snijder, Marjolein Kikkert
bioRxiv 751578; doi: https://doi.org/10.1101/751578
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The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model
Robert C. M. Knaap, Raúl Fernández-Delgado, Tim J. Dalebout, Nadia Oreshkova, Peter J. Bredenbeek, Luis Enjuanes, Isabel Sola, Eric J. Snijder, Marjolein Kikkert
bioRxiv 751578; doi: https://doi.org/10.1101/751578

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